Use of the human lrp/mvp promotor for a vector that can be induced by therapy

ABSTRACT

The subject matter of the invention is a therapy-regulatable vector system of the gene promoter of the human lung resistance protein (LRP/MVP). This vector system for the therapy-inducible expression of therapeutically relevant genes in mammalian cells shall be used in the pharmaceutical industry and in medicine.  
     The invention has the objective of regulating the tumor gene therapy through therapeutic modalities, such as chemotherapy or hyperthermia, for the period of these therapies, so that the gene therapy, together with chemotherapy, hyperthermia, and other means, results in a controlled, more efficient treatment of the tumor disease.  
     The essential characteristic of the invention is a therapy-inducible vector for expressing therapeutic genes on the basis of vector base structures that are suitable for the expression in mammalian cells. It is characterized by a human LRP/MVP promoter or a defined sequence thereof, which is inducible by chemotherapy, hyperthermia, and other means, and a gene encoding for a therapeutic protein or non-translated RNA.

SUBJECT MATTER OF THE INVENTION

[0001] The subject matter of the invention is a therapy-regulatable vector system of the gene promoter of the human lung resistance protein (LRP/MVP). This vector system for the therapy-inducible expression of therapeutically relevant genes in mammalian cells is to be used in the pharmaceutical industry and in medicine.

SCIENTIFIC BACKGROUND AND DESCRIPTION OF INVENTION

[0002] Increasing, gene-therapy strategies are pursued for the treatment of tumor diseases. It is hereby often of interest to express the therapeutic gene for a defined period of time in a sufficient quantity in the target cells and let it become effective for tumor therapy. For this reason, promoters that are inducible by way of defined, therapy-associated modalities, are important for the construction of conditionally active vectors in gene therapy. Vectors that can be regulated by a specific therapy enable a controlled expression of the therapeutic gene (Walther 1996, Mol. Biotechnol. 6: 267-86).

[0003] The invention has the objective of regulating the tumor gene therapy through therapeutic modalities, such as chemotherapy or hyperthermia, for the period of these therapies, so that the gene therapy, together with chemotherapy, hyperthermia, and other means, results in a controlled, more efficient treatment of the tumor disease.

[0004] The objective of the invention is realized by using an expression vector according to claim 1. Secondary claims 2-8 characterize the vector according to the invention in more detail. An essential characteristic is a vector that is characterized by a therapy-inducible LRP/MVP promoter or a defined sequence thereof and by a gene encoding for a therapeutically relevant protein. This promoter is inducible by chemotherapy, hyperthermia, and other means, and in this way permits the therapy-induced expression of subsequent therapeutic genes.

[0005] It was unexpectedly found that the human LRP/MVP gene promoter isolated and cloned by the inventors is inducible through therapy-relevant factors, and the expression of subsequent genes thus can be controlled. These inductors include chemotherapy agents, such as cisplatin, 5-fluoruracil, adriamycin, vincristin etc., but also other therapy-associated factors, such as, for example, hyperthermia and irradiation. The vector of the invention according to claim 1 therefore enables the cytostatic- and/or hyperthermia-induced expression of therapeutic genes in the target cells (tumor cells), and therefore a combination of gene therapy and chemotherapy, hyperthermia, and/or irradiation.

[0006] As a base structure for the vectors according to the invention, all constructs suitable for expression in mammalian cells can be used. These include vector base structures based on DNA (for example, adenoviruses, AAV) or RNA viral vectors (for example, MoMuLV, HIV). The vectors according to the invention therefore can be introduced into the target cells with suitable non-viral (gene gun, liposomes, electroporation) or viral carrier systems and unfold the therapeutic effect. The therapeutic effect of the vector according to the invention is created in that, after the vector transfer by chemotherapy, hyperthermia, or irradiation, the expression of the therapeutic gene is induced by the LRP/MVP promoter, and the resulting antitumor gene products are released in the tumor cell and, as the case may be, in its environment. With the help of the invention, the combination of gene therapy (antitumor effects of the induced therapeutic genes) and chemotherapy, hyperthermia, and/or irradiation is able to result in improved tumor therapy.

[0007] The invention is described in more detail using the following embodiments:

EXEMPLARY EMBODIMENT 1 Cloning of Human LRP/MVP Gene Promoter

[0008] Isolation of the Human LRP/MVP Promoter

[0009] From the human, multidrug-resistant, but non-P-glycoprotein-expressing lung cancer cell line SW1573, we isolated a 5′ upstream sequence of the LRP gene with a size of approximately 1.9 kb (claim 1). For this, the genomic DNA was isolated from SW1573 cells, and the genome walking method was used in different PCR versions. After cloning the approximately 1.9 kb 5′ flanking sequence of the LRP gene, several consensus elements for the binding of transcription factors were identified after the sequence analysis: for example, an inverted CCMT box (Y-box) for binding the transcription factor YB-1 described in the context with resistance, an E-box for binding c-myc, as well as sequences for binding p53 and SP1.

[0010] Identification of the Transcription Start

[0011] To identify the transcription start, we constructed a cDNA library of the SW1573 cell line using RACE-PCR (rapid amplification of cDNA ends). We hereby found an alternative 3′ splicing site in intron 1 of the LRP gene. This alternative splicing within the 5′ untranslated region in intron 1 then results in two different versions of LRP-mRNA.

[0012] Detection of Promoter Activity

[0013] To analyze the promoter activity, the following constructs were produced and tested with a chloramphenicol acetyl transferase (CAT) assay: pCAT-MVPprom1.9s (includes the 1.9 kb sequence in sense orientation under control of the CAT promoter), pCAT-MVPprom1.9 as (in antisense orientation), pCAT-MVPprom0.7s (includes a 0.7 kb deletion version of the 1.9 kb sequence), and pCAT-MVPprom0.7 as. The pCAT basic (without promoter) and pCAT control constructs (with SV40 promoter) as well as the mentioned LRP promoter each were transfected into two human tumor cell lines, and a reporter gene assay (CAT-ELISA) was performed. Promoter activities were detected for both LRP promoter constructs, whereby the one for pCAT-MVPprom0.7s was unexpectedly higher than the one for pCAT-MVPprom1.9s and pCAT control (FIG. 1).

[0014] Detection of Inducibility via Cytostatics

[0015] Beyond the basal promoter activity, the LRP/MVP-promoter-mediated expression of subsequent genes can be induced via a number of cytostatics. Below, the 5-fluoruracil-induced CAT expression is shown as an example: (FIG. 2)

EXEMPLARY EMBODIMENT 2 Construction of the Therapy-Inducible Vector Construct Using the LRP/MVP Gene Promoter

[0016] Constructions of Therapy-Inducible Vectors for Expressing Therapeutic Genes

[0017] The LRP/MVP promoter or parts thereof are inserted into vector base structures in such a way that the promoter regulates the expression of a subsequent therapeutic gene. Such vector base structures may be non-viral expression vectors (for example, pcDNA3, pcDNA6, etc.) or viral vector base structures (for example, adenoviral vectors, retroviral vectors pLXSN, pLXIN, etc.).

[0018] Gene Transfer of Vector Constructs

[0019] The transfer of the vector constructs may take place with the help of non-viral transfer technologies. Such technologies may be the vector injection, in vivo electroporation, particle bombardment, needle injection, or liposomal transfer (Li, 2000, Gene Ther. 7: 31-4). Alternatively, for viral vector constructs, viral gene transfer can be used for the transfer into the target cells by way of producing recombinant DNA or RNA virus particles with the help of suitable helper cell lines (Walther, 2000, Drugs 60: 249-71). These technologies are already being used clinically or are used prospectively for treating cancer patients.

LEGENDS FOR FIGURES

[0020]FIG. 1

[0021] Detection of LRP/MVP promoter activity in CAT assay in human colon cancer cell lines HCT116 (black) and HCT15 (white).

[0022]FIG. 2

[0023] Detection of inducibility of LRP/MVP promoter by 5-fluoruracil (black) 24 h after cytostatic administration. 

1. Therapy-inducible vector for expressing therapeutic genes based on vector base structures that are suitable for the expression in mammalian cells, characterized by a human LRP/MVP promoter inducible by chemotherapy or hyperthermia or defined sequence thereof with the same properties and a gene encoding for a therapeutic protein or non-translated RNA.
 2. Therapy-inducible expression vector according to claim 1, characterized by the following sequence of the human LRP/MVP promoter or parts thereof with the same properties (numbering starting at transcription start): −1859 GGTACCTGCAGTGGCATGATCTTGGCTCACTGCAATCTCCACCTCCTGGGTTGAAGCGATTCTCCTACCTCAGTCTCCTGAGTAGCTGGGATTAC −1764 AAGGACACGTCACCACGCCTAGCTAATTTTTGTATTTTCTAGTAGAGAGGGGGTTTCGCCATGTTGGCGAGGCTGGTCTTGAACTCCTGACCTAA −1669 AGCTTCCCAAAGTGCCTGCCTCAGCTTCCCAAAGTGCTGGAATTACAGGCGTGAGCTGCTGCACTCAGCCATAAATCGTGTCACTTTTCCACTTA −1574 AAATTTTCCAAGGGATTCCATCTTGCCAGGGAAGAGCATGTCAAAGGAGAATCCAAGCGCTTTTCCCGCCACCTCCAGTTCTCTGCACTCTTTTT −1479 TTTGTTTTTGTTTTTGGCGAGTAGGGGAGACGGAGTGTAGCTGTGTCACCGAGGCTGGAGTGCAGTAGTGCGATCTTGGCTCATTGCAACCTCCA −1384 CCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTTCCAAGTAGCTGGGATTACAGGCGCCCACCACCACGCCCAGCTAATTTTTTGTGTTGGCC −1289 AGGCTGGACTCAAACTCCTGACCTCGAGAGTTGCCCACCTCAGCCTCCCAAAGTGCTGGCATTACAAGCGTGAACCACCATGCCCGGCCTGCACT −1194 CTTCTTTGAACAGAACTCTGTTCTTGTCCTGGGGCCTATACCCTTGCCATTCCCCTGCCCAGTATGTTCCTCCCTGTTCTTCACATTACCTGTGC −1099 CTTCCTGTCAATCAAGATCTTTGCCTCTCACCCTCTCTGAGGTCCAACCCTGAACCTCAGGTCCTCCAAGACAGAGGCCTGGGGACCTGCATGTT −1004 TAACCAGCTCCCCAAGTGATGAGTGAGGTCCAGGCAGGTATGGTGTACACCACCTCCATCCCTTCCATGTATCTTACCTTCCTCTTCTCCAGGAA −909 GCTCAGCCCGGAGCCAGAAACGGGAGGCCCGCCTGGACAAGGTGCTGTCGGACATGAAGAGACACAAGAAGCTGGAGGAGCAGATCCTTCGTACC −814 GGGAGGGACCTCTTCAGCCTGGACTCGGAGGACCCCAGCCCCGCCAGCCCCCCACTCCGATCCTCCGGGAGTAGTCTCTTCCCTCGGCAGCGGAA −719 ATACTGATTCCCACTGCTCCTGCCTCTAGGGTGCAGTGTCCGTACCTGCTGGAGCCTGGGCCCTCCTTCCCCAGCCCAGACATTGAGAAACTTGG −624 GAAGAAGAGAGAAACCTCAAGCTCCCAAACAGCACGTTGCGGGAAAGAGGAAGAGAGAGTGTGAGTGTGTGTGTGTGTTTTTTCTATTGAACACC −529 TGTAGAGTGTGTGTGTGTGTTTTCTATTGAACACCTATAGAGAGAGTGTGTGTGTTTTCTATTGAACATCTATATAGAGAGAGTGTGTGAGTGTG −434 TGTTTTCTATTGAACACCTATTCAGAGACCTGGACTGAATTTTCTGAGTCTGAAATAAAAGATGCAGAGCTATCATCTCTTAAAAGGAGGGGCTG −339 TAGCTGTAGCTCAACAGTTAGGCCCCACTTGAAGGGAGAGGCAGAATTGTACTCACCCAGATTGGAAAATGAAAGCCAGATGGGTAGAGGTGCCC −244 TCAGTTAGCACCTGTCCCATCTCGGGCCCTCCAACTCCTCCCAGTCCCACTCCAGTGCAGCCAGCTGGCTCCAAGGTAGAAACCCATGAGCACTC −149 AGGGAGCAGTGTGCCTTCAGCTGCAGCAGAAGCAGCCCGGAGGATAAAATGAGAACCAGCTGCACACGGGCCCTTTAACTCCCAAGCCCCACCCC −54 TGGGCTTGGCCTGCCTTGCCCTGCCGGGAAGTGATCCCCAAGGCAGGGTGAGAGTTCCCCATCTGAGGCGTTTGTTGCAGCTACCTGCACTTCTA +33 GAT


3. Therapy-inducible expression vector according to claim 1 and 2, characterized by mutation versions, such as, for example, insertions, deletions, and/or substitutions with the same properties.
 4. Therapy-inducible expression vector according to claim 1-3, characterized by a gene of a cytokine, enzyme, antibody, apoptosis gene, resistance gene, anti-oncogene, or non-translated RNA.
 5. Therapy-inducible expression vector according to claim 1-4, characterized by the gene of a cytokine, such as, for example, tumor necrosis factor alpha, interferon alpha and gamma, interleukin 2, 6, 7, 12, colony stimulating factors GM-CSF or G-CSF.
 6. Therapy-inducible expression vector according to claim 1-5, characterized by the gene of an enzyme, such as that of herpes simplex thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P-450.
 7. Therapy-inducible expression vector according to claim 1-6 for producing a therapeutic agent against tumor diseases, characterized by an expression vector for a non-viral gene transfer.
 8. Therapy-inducible expression vector according to claim 1-6 for producing a therapeutic agent against tumor diseases, whereby these, as the case may be, may be packaged in viral particles for the viral gene transfer. 